Feature Articles

Maximizing HPLC Power and Throughput

Technology Heats Up

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2-D LC of BSA tryptic peptides: Peak capacity of ~1,100 at a rate of 1 peak/second [Peter Carr, Ph.D., University of Minnesota]

In May, Waters announced a partnership with NIBRT focused on creating a database for glycan analysis based on the company’s UltraPerformance Liquid Chromatography® (UPLC®) platform. NIBRT will develop, maintain, and license the database, which should launch in 2011 and will be co-marketed by NIBRT and Waters.

The database will contain chromatographic retention times for sets of glycan structures associated with biotherapeutic compounds. It is intended for use by biopharmaceutical manufacturers as a tool for evaluating the structure of glycosylated proteins and as a means of monitoring product integrity and bioprocess control. Researchers will use the Acquity UPLC system with an ethylene bridged hybrid glycan separation column and fluorescence detection to separate the glycans released from glycoproteins in the form of 2-aminobenzamide derivatives.

Peter Carr, Ph.D., professor in the department of chemistry at the University of Minnesota, Minneapolis, describes three main technological advances in HPLC “that have come together to push it forward”: the instrumentation that enabled ultrahigh pressure LC (UHPLC), which was based on the work of Jim Jorgenson, professor in the department of chemistry at the University of North Carolina, Chapel Hill; the development of micropellicular particles, composed of a solid inner core that is chromatographically inactive and impermeable, surrounded by a thin crust of porous material that is chromatographically active; and the use of high temperatures, which has been the focus of Dr. Carr’s research.

Dr. Carr describes temperature as “the third dimension in HPLC.” He is using higher temperatures to decrease fluid velocity, which yields an increase in the flow rate without the need for higher pressures and raises the diffusion coefficients to enhance mass transfer and limit peak broadening.

To achieve the benefits of increased temperature without compromising the quality of the chromatographic separation, Dr. Carr’s group has developed thermally stable stationary phases—zirconia-based and hyper-crosslinked silica-based phases. They are using these media to perform ultrafast high-temperature LC (UFHTLC) as the second dimension in comprehensive 2-D LC/LC for analytical applications, in which all of the material that exits the first column goes into the second column.

The increased speed enabled by the high temperatures is not only necessary to handle the large number of samples produced by the first-dimension LC separation, but it also provides another, unexpected benefit. “As you do the second dimension faster, your overall resolving power improves,” says Dr. Carr. If the speed of the second dimension is less than optimal and, consequently, “you don’t take enough fractions out of the first dimension,” the fractions will be too big, remixing can occur, and “you lose information that is already there.”

By running the second dimension at optimal speed, which Dr. Carr estimates from experimental data as a separation every 15–20 seconds, it is possible to achieve maximal peak capacity.

In 2-D LC/LC, the cycle time of the second dimension has a greater impact on the overall 2-D peak capacity than does the first dimension, explains Dr. Carr, and he recommends the use of strategies such as higher temperatures to optimize the speed of the second dimension rather than options such as microparticle-based media combined with high pressures, or long monolithic columns, to accelerate the first-dimension separation.

Core-Based Particle Design

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The first major advance in faster HPLC led to the development of UHPLC instrumentation, marked by the introduction of Waters’ Acquity UPLC technology for high-speed chromatography in 2004. UHPLC systems, which include Agilent Technologies’ Infinity LC and 1200 Series Rapid Resolution LC (RRLC), and Shimadzu Scientific Instruments’ Prominence UFLC, achieve faster analysis speeds without loss of chromatographic fidelity.

The combination of UHPLC technology and sub-2 micron particles maximized the efficiency of these systems. Agilent, for example developed the Zorbax Rapid Resolution HT columns, which are packed with porous 1.8-micron particles; the Zorbax line includes the company’s 300StableBond wide-bore 300Å columns.

The third main advance described by Dr. Carr was the development of smaller pellicular particles that offer a short diffusion distance and the combined benefits of the pressure drop created by a 2.8-micron particle and the narrow peaks achieved with sub-2 micron particles. Compared to sub-2 micron particles, the pellicular particles do not require high back pressures and can be used on conventional HPLC instruments.

Advanced Materials Technology’s Halo™ media is based on the company’s Fused-Core™ technology. Sigma-Aldrich recently added the Peptide ES-C18 column, based on a 160Å fused-core particle design, to its line of Ascentis® Express columns. Agilent offers its Poroshell columns with particles containing a solid silica core and porous outer shell.

Phenomenex introduced its 2.6 micron Kinetex core shell product in August 2009. The particle contains a 1.9-micron solid core and a 0.35-micron porous outer layer and offers pressure stability up to 600 Bar in 4.6 mm internal diameter columns and up to 1,000 Bar in 2.1 mm internal diameter columns. The company’s 1.7-micron core shell particle has a 1.25-micron core and a 0.23-micron shell and provides pressure stability up to 1,000 Bar. Both particles are available as C18, pentafluorophenyl (PFP, for separating isomeric and halogenated compounds), and hydrophilic interaction LC (HILIC) columns.

“If a customer is using a 5-micron particle, switching to Kinetex provides a threefold increase in efficiency; switching from a 3-micron particle provides twice the efficiency,” says Terrell Mathews, head of product management at Phenomenex. Many customers are using the core shell particles not only to increase throughput, “but to increase chromatographic resolution and sensitivity.”

The core shell technology “will be our platform for future development,” Mathews says. The company plans to develop new phases and new chemistries based on these particles. Customers are asking for a range of larger particle sizes, additional chemistries, and a selection of different column dimensions, he notes.

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